The infected cell 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 -conjugating enzyme, and possesses in vitro E3 activity

Charles Van Sant, Ryan Hagglund, Pascal Lopez, and Bernard Roizman†

The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, 910 East 58th Street, Chicago, IL 60637

Contributed by Bernard Roizman, June 5, 2001 The infected cell protein 0 (ICP0) of herpes simplex virus 1, a led us to investigate the possibility that ICP0 expresses this promiscuous transactivator shown to enhance the expression of function. In addition, it has been reported that foci of ICP0 introduced into cells by infection or transfection, interacts observed in infected and transfected cells contain enhanced with numerous cellular and has been linked to the dis- levels of conjugated ubiquitin, which is consistent with E3 ligase ruption of ND10 and degradation of several proteins. ICP0 contains activity (21, 22). a RING finger domain characteristic of a class of E3 ubiquitin Relevant to this report are the following: (i) In the R7914 ligases. We report that: (i) in infected cells, ICP0 interacts dynam- recombinant virus, the aspartate 199 of ICP0 was replaced with ically with proteasomes and is bound to proteasomes in the alanine. This substitution abolished the binding cyclin D3 and presence of the proteasome inhibitor MG132. Also in infected cells, precluded the colocalization of cyclin D3 with promyelocytic cdc34, a polyubiquitinated E2 ubiquitin-conjugating enzyme, ex- leukemia protein and the transport of ICP0 to the cytoplasm but hibits increased ICP0-dependent dynamic interaction with protea- did not preclude the degradation of ND10 structures (23). The somes. (ii)Inanin vitro substrate-independent ubiquitination R7914 mutant virus carrying the ICP0 D199A substitution system, the RING finger domain encoded by exon 2 of ICP0 binds exhibits a 10-fold reduction in viral yields from quiescent HEL cdc34, whereas the carboxyl-terminal domain of ICP0 functions as fibroblasts and reduced neuroinvasiveness (24). an E3 ligase independent of the RING finger domain. The results (ii) Ubiquitin is activated by the E1 ubiquitin-activating en- indicate that ICP0 can act as a unimolecular E3 ubiquitin ligase and zyme in an ATP-dependent manner to form a thioester with a that it promotes ubiquitin-protein ligation and binds the E2 cdc34. conserved active site cysteine. The major E1 in both yeast and It differs from other unimolecular E3 ligases in that the domain humans is Uba1. Activated ubiquitin is transesterified to a containing the RING finger binds E2, whereas the ligase activity conserved cysteine of an E2 ubiquitin-conjugating enzyme. maps to a different domain of the protein. The results also suggest There are 13 E2s identified in yeast, and mammals have at least that ICP0 shuttles between nucleus and cytoplasm as a function of that number. The E3 ubiquitin ligase binds both the E2 and its dynamic interactions with proteasomes. substrate and functions to assemble ubiquitin onto the substrate. E3s are by far the most diverse components of the system nfected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) (reviewed in ref. 25). A recently described ubiquitination enzyme Iacts as a promiscuous transactivator of viral and cellular genes is the E4 multiubiquitin chain assembly factor, which binds (reviewed in ref. 1). ICP0 is critical for viral replication in cells ubiquitin moieties conjugated to the substrate and drives the MICROBIOLOGY infected at low multiplicity but is not essential in cells infected polymerization of long polyubiquitin chains (26). This mecha- at high multiplicity (2, 3). In euploid human embryonic lung nism facilitates targeting of the substrate for degradation by the (HEL) fibroblasts, ICP0 is transported into the cytoplasm be- 26S proteasome. tween5and7hafterinfection. The protein localizes with the (iii) A substrate-independent in vitro ubiquitination system can promyelocytic leukemia protein, a component of a nuclear be constructed by addition of recombinant ubiquitin-activating structure known as ND10 (4), and causes its disruption (5–7). It (E1) enzyme, recombinant ubiquitin-conjugating (E2 or Ubc) also interacts with several proteins such as the BMAL1 trans- enzyme, recombinant ubiquitin ligase (E3) enzyme, ubiquitin, activator (8), the translation elongation factor 1␦ (9), cyclin D3 and ATP. Here we report that a domain of the ICP0 encoded by (10), and a ubiquitin-specific protease USP7 (11–13). exon 3 substitutes for and acts as an E3 ubiquitin ligase, whereas Several lines of investigation have led to the suggestion that the domain encoded by exon 2 containing the RING finger binds ICP0 also interacts with the ubiquitin-proteasomal degradation a E2 protein. pathway. The evidence includes the association with USP7 and the functional association with the degradation of sumoylated Materials and Methods promyelocytic leukemia protein and other as-yet-unidentified Low passage HEL fibroblasts grown in 150-cm2 flasks were sumoylated proteins (14), the regulatory and catalytic subunits exposed to 10 plaque-forming units (pfu) of HSV-1(F) (27) or to of DNA-dependent protein kinase (15, 16), centromeric proteins the R7914 recombinant virus described elsewhere (24, 28). After C and A (17, 18), and Sp100 (19, 20). In addition, this laboratory demonstrated that ICP0 is dynamically associated with protea- Abbreviations: ICP0, infected cell protein 0; HSV-1, herpes simplex virus 1; HEL, human somes in untreated cells but remains bound to proteasomes in embryonic lung; pfu, plaque-forming unit; GST, glutathione S-transferase; SCF, Skp1- cells treated with proteasomal inhibitor MG132. Last, the 775-aa Cdc53-F-box protein. ICP0 is translated from a spliced mRNA. The three exons †To whom reprint requests should be addressed. E-mail: [email protected]. encode 19, 222, and 534 codons, respectively. A RING finger The publication costs of this article were defrayed in part by page charge payment. This domain characteristic of E3 ubiquitin ligases has been identified article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. in the domain encoded by exon 2. These properties of ICP0 have §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.161283098 PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8815–8820 Downloaded by guest on October 1, 2021 2 h of exposure, the inoculum was replaced with conditioned spent medium and incubated at 37°C.

Immunoprecipitation and Pulldown Experiments. Infected or mock- infected cells were harvested, pelleted by centrifugation, and solubilized at 4°C in modified proteasome immunoprecipitation buffer [0.5% Nonidet P-40͞50 mM Tris, pH 7.5͞12% glycerol͞ 0.1% Na orthovanadate͞10 mM NaF͞0.5 mM DTT͞2.5 mM ͞ ͞ MgCl2 l-1-tosylamido-2-phenylethyl chloromethyl ketone 7- amino-1-chloro-3-tosylamido-2-heptanone͞PMSF͞an ATP re- generating system consisting of 0.2 mM ATP͞1 mM creatine phosphate͞15 units of creatine phosphokinase (29)]. After a brief sonication, lysates were clarified by centrifugation at 2,000 ϫ g at 4°C and reacted with either normal mouse or rabbit Fig. 1. Antibody to ICP0 pulls down the HC8 proteasome subunit from cells sera for 1 h, precleared by mixing with 5 mg each of protein-G infected with wild-type virus and treated with MG132. HEL fibroblasts in- and protein-A Sepharose, and then reacted overnight at 4°C with fected with 10 pfu of HSV-1(F) per cell were mock-treated or treated for 1 h either 2 ␮g of mouse monoclonal antiserum to proteasome before harvest at 5, 10, or 18 h after infection, lysed, and reacted with polyclonal rabbit antibody against ICP0-exon 2. The precipitate was solubi- subunit XAPC7 (␣4, Affiniti catalogue no. PW8120), 5 ␮lof ␮ lized, subjected to electrophoresis in a denaturing gel, and reacted with rabbit polyclonal anti-HSV thymidine kinase or 2 l of rabbit mouse monoclonal antibody against HC8. anti-ICP0 exon 2 antibody (10). Each sample was then reacted with a mixture of protein-G Sepharose and protein-A Sepharose (5 mg each) for 1 h. The Sepharose pellet was collected, rinsed Mouse monoclonal antibody against ICP0, Goodwin 1112 extensively with proteasome immunoprecipitation buffer, and (Goodwin Institute, Plantation, FL; ref. 30), was diluted at ͞ then solubilized in disruption buffer (2% SDS 50 mM Tris, pH 1:2,000. Rabbit polyclonal antiserum was diluted in antibody ͞ ͞ ␤ 6.8 3% sucrose 5% -mercaptoethanol/bromophenol blue). diluent as follows: ICP0 exon 2 (10) 1:1,000, Ccdc34 (Neomar- kers Freemont catalogue no. RB-043-P1) 1:500, and GST-ORF In Vitro Substrate-Independent Polyubiquitination. Thirty microli- P (31) 1:2,000. Immunoblots for ICP0 and GST were rinsed in ters of in vitro reactions were performed in ubiquitination buffer ͞ ͞ PBS containing 0.1% Tween-20 and both reacted to secondary [50 mM Tris, pH 7.5 2.5 mM MgCl 0.5 mM DTT] and con- antibody [alkaline phosphatase-conjugated goat anti-rabbit tained 40 ng of recombinant E1 (Calbiochem catalogue no. (1:3,000), Sigma] for 2 h. Immunoblots for cdc34 and HC8 were 662070), 40 ng [His ]-UbcH3 (Affiniti, Mamhead, Exeter, U.K., 6 rinsed and reacted to secondary antibody [peroxidase- catalogue no. UW8730), 2 ␮g of biotinylated ubiquitin (Affiniti catalogue no. UW8705), and 0.2 mM ATP along with the ATP conjugated goat anti-rabbit and anti-mouse (1:1,000), Sigma], regenerating system described above where indicated. Reaction respectively, for 2 h. Immunoblots were developed by either mixtures also contained 5 ␮g of purified glutathione S- enhanced chemiluminescence (SuperSignal West Pico chemilu- transferase (GST), GST-ICP0 exon 2 (pRB4994), or GST-ICP0 minescent substrate, Pierce) or 5-bromo-4-chloro-3-indolyl ͞ exon 3 (pRB4995), previously described (9). The mixtures were phosphate nitroblue tetrazolium substrate (Sigma), according allowed to react at 37°C for 90 min. The reaction was either to instructions supplied by the manufacturer. stopped by the addition of disruption buffer or subjected to affinity capture. Results Affinity capture was done with either glutathione-Sepharose Wild-Type ICP0 Associates with Proteasome Complexes in Infected (Sigma) or Talon metal affinity resin (CLONTECH). Each Cells. In these experiments, ICP0 was immune precipitated from sample (15 ␮l) from in vitro ubiquitination reactions described lysates of HEL fibroblasts that were mock-treated or treated with above was diluted on wet ice in 500 ␮l of ubiquitination buffer, MG132 at various times after exposure to 10 pfu of HSV-1(F) mixed with either 1 mg of glutathione-Sepharose or 20 ␮lof per cell. Electrophoretically separated immune precipitates were Talon resin, and kept for1hat4°C. The pellets were rinsed then probed with antibody to the proteasome subunit HC8 (␣7). extensively with ubiquitination buffer before addition of disrup- The procedures were as described in Materials and Methods. The tion buffer. results shown in Fig. 1 indicate that HC8 coprecipitated with ICP0 from lysates of MG132-treated infected cells but not from Immunoblotting. Cells were rinsed in PBS containing an EDTA- untreated infected cells (compare lanes 4 and 5, 6 and 7, and 8 free protease inhibitor mixture (Roche Diagnostics) and lysed in and 9). The amount of HC8 that coprecipitated with ICP0 PBS-A* (1% Nonidet P-40, 1% deoxycholate in PBS containing increased as infection progressed (compare lanes 5, 7, and 9). In protease inhibitor). Cell lysates, affinity captured proteins, or other experiments, proteasome subunits HC3 (␣2) and XAPC7 immune precipitates were solubilized in disruption buffer, boiled (␣4) also coprecipitated with ICP0 (Fig. 3B and data not shown). for 5 min, subjected to electrophoresis on 10% N,NЈ- To control for nonspecific association of ICP0 with immune diallyltartardiamide-acrylamide gels, and transferred to nitro- complexes, we precipitated viral thymidine kinase protein with cellulose sheets. For detection of biotinylated ubiquitin, the nitrocellulose sheets were blocked for1hwithPBSsupple- polyclonal antibody to the protein from lysates of HEL cells mented with 0.1% BSA and 0.1% Tween-20. After blocking, the harvested at5hafterinfection, as described above. The elec- membrane was reacted for1hwithstreptavidin-peroxidase trophoretically separated precipitates were probed with antibody (1:1,000 dilution, Bio-Rad) in blocking buffer, extensively rinsed, to ICP0. The results were that ICP0 was present in cell lysates but and developed by enhanced chemiluminescence according to was not coprecipitated with the viral thymidine kinase (data not instructions supplied by the manufacturer (Pierce). For all other shown). immunoblots, the membranes were blocked for1hwith5% Inasmuch as ICP0 interacted with three different proteasomal nonfat dry milk and reacted with the appropriate primary subunits in the presence of MG132, but not in the absence, of the antibody overnight at 4°C. Monoclonal antibody to proteasome, drug, we conclude that ICP0 interacts dynamically with protea- subunit ␣7 (HC8) (Affiniti catalogue no. PW8110), was diluted somes and that, in the presence of MG132, ICP0 remains 1:1,000 in PBS supplemented with 1% BSA and 0.1% Tween-20. associated with proteasomes.

8816 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.161283098 Van Sant et al. Downloaded by guest on October 1, 2021 mixture was electrophoretically separated in a denaturing poly- acrylamide gel and reacted with streptavidin to identified ubi- quitinated species. Fig. 3B shows an identical copy of the electrophoretically separated polypeptides reacted with antibody to GST. In a parallel experiment, the His6-cdc34 was collected by affinity chromatography, electrophoretically separated in a replicate denaturing polyacrylamide gel, and reacted with streptavidin. This procedure enabled the detection of ubiquiti- nated species interacting with cdc34 or ubiquitinated cdc34. The results were as follows: (i) Significantly higher levels of ubiquitinated products and higher molecular weight ubiquiti- nated forms were observed in reactions containing GST-exon 3 in the presence of ATP (Fig. 3A, lane 6) than in any other reactions, suggesting that ICP0-exon 3 promotes ubiquitin- protein ligation. The observation that some bands in the ladder differ by approximately MR of 9,000 is consistent with the Fig. 2. Monoclonal antibody to XAPC7 pulls down cdc34 from lysates of HEL ligation of integral numbers of biotinylated ubiquitin molecules. fibroblasts infected with wild-type HSV-1(F) and treated with MG132. Repli- This property is characteristic of E3 activity in substrate- cate cultures of HEL fibroblasts were infected with 10 pfu or HSV-1(F) or R7914 independent in vitro systems (reviewed in ref. 25). mutant virus per cell and either left untreated or treated with MG132 for 1 h (ii) Comparison of lanes 5 and 6 in Fig. 3A indicates that the before harvest at 9 h after infection. The immune precipitates obtained with ubiquitination reaction and especially the appearance of higher monoclonal antibody against XAPC7 proteasome subunit were electro- molecular weight ubiquitinated proteins observed in lane 6 of phoretically separated in denaturing gels and reacted with polyclonal rabbit Fig. 3A were ATP-dependent. This would be expected, because serum made against cdc34. the ubiquitin-protein ligation is ATP dependent. Therefore, the ubiquitinated products are the consequence of the cascade of ICP0 Enhances a Dynamic Association of Ubiquitinated cdc34 with ubiquitinating enzymes and not an artifact of the system, because Proteasomes. HEL cells were exposed to 10 pfu of HSV-1(F) or charging of ubiquitin by E1 is ATP dependent (34). (iii) GST (Fig. 3A, lane 2), other GST fusion proteins (data not R7914 per cell and were either untreated or treated with 5 ␮m shown), or E1 and E2 without a putative E3 (Fig. 3A lane 7) MG132 for 1 h prior to harvest at9hafterinfection. Lysates failed to promote ubiquitin polymerization. Although ATP- prepared in proteasome precipitation buffer supplemented with dependent ubiquitin ligation was observed in the in vitro system ATP were reacted with antibody to XAPC7. The immune in the absence of GST-exon 3, it occurred at much lower levels precipitate was electrophoretically separated in a denaturing (compare lanes 2, 4, and 7 with lane 6), and high molecular polyacrylamide gel and probed with antibody to cdc34 (UbcH3), weight ubiquitinated forms were not observed. This observation the E2 enzyme that interacts with Skp1-Cdc53-F-box protein can be explained by basal ubiquitin-protein ligation activity in (SCF) (reviewed in ref. 25). As shown in Fig. 2, lane 3, the the in vitro system mediated by the E2 in the absence of immune precipitate from lysates of MG132 treated wild-type stimulation by an E3. virus-infected cells contained several cdc34 isoforms that Ͼ (iv) Fig. 3B shows that the chimeric proteins GST-exon 2 and formed bands with a MR 50,000. These isoforms did not GST-exon 3 were comparable in amount and of predicted precipitate with proteasomal components from lysates of un- molecular weight. Therefore the difference in E3 activity be- treated wild-type virus-infected cells or of cells infected with tween GST-exon 3 and GST-exon 2 cannot be attributed to R7914 recombinant virus, although small amounts of high differential addition of the GST fusion proteins to the reactions. molecular weight protein were present in all precipitates. (v) Multiple ubiquitinated forms of cdc34 were observed in The observation that cdc34 [MR of 32,000 (32)] coprecipitat- reactions containing GST-exon 3 but not in reactions containing MICROBIOLOGY ing with proteasome complexes migrated more slowly than GST, GST-exon 2, or no GST fusion protein (Fig. 3C). Obser- expected suggests that it was covalently modified with numerous vations that some bands in the ladder differed by approximately ubiquitin adducts. This conclusion is likely to be correct, inas- Mr of 9,000 and that there were protein bands with an Mr below much as, in addition to being charged at cysteine, yeast cdc34 is the expected size (Mr of 41,000) for monoubiquitinated cdc34 are ubiquitinated at multiple lysines (33). Furthermore, because the consistent with the ligation of integral numbers of ubiquitin higher MR cdc34 isoforms were present in significantly higher molecules to cdc34. This observation suggests that ICP0-exon 3 amounts in proteasomes precipitated from lysates of wild-type promotes autoubiquitination of the E2, cdc34. Several bands in virus-infected cells treated with MG132 but not from those of this ladder (Fig. 3C, lane 17) correspond to bands in the blot mock-treated infected cells or cells infected with R7914 mutant, showing total ubiquitinated species (Fig. 3A, lane 6), suggesting the results suggest that the enhanced interaction of ubiquitinated that autoubiquitination of cdc34 accounts for a significant cdc34 with proteasomes is dynamic and mediated by wild-type amount of the ubiquitin-protein ligation promoted by ICP0 exon ICP0. 3 in the substrate independent in vitro ubiquitination system. In such systems, E3 activity often promotes autouibiquitination of ICP0 Expresses an E3 Ubiquitin Ligase Function. In current models of the E2 (reviewed in ref. 25). the ubiquitin-proteasomal pathway, E3 ubiquitin ligases couple (vi) In substrate-independent in vitro ubiquitination systems, with E2 to bind substrate and facilitate the assembly of a some RING finger E3 ligases were ubiquitinated (35). In this polyubiquitin chain on the substrate (reviewed in ref. 25). system, ICP0 exon 3 was not ubiquitinated to a significant extent Because ICP0 possesses a RING finger characteristic of many E3 (data not shown). enzymes, we used an in vitro ubiquitination system to determine These results indicate that ICP0 has E3 ubiquitin ligase whether the ICP0 chimeric proteins GST-exon 2 (ICP0 amino activity that maps to the region between amino acids 568 and 773 acids 111–240) or GST-exon 3 (ICP0 amino acids 568–773) in the C-terminal portion of exon 3. promoted substrate-independent ubiquitin-protein ligation in the system described in Materials and Methods. In the experi- ICP0 Interacts with the E2 Ubiquitin-Conjugating Enzyme cdc34. The ment, the results of which are shown in Fig. 3A, the reaction results presented in Fig. 2 indicated a dynamic interaction of

Van Sant et al. PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8817 Downloaded by guest on October 1, 2021 Fig. 3. The domain encoded by the carboxyl terminus of exon 3 of ICP0 acts as an E3 ligase, whereas the sequences encoded by exon 2 bind cdc34 ubiquitin-conjugating enzyme. (A) Immunoblots of electrophoretically separated products of substrate-independent in vitro ubiquitination reactions. GST (lanes 1 and 2), GST-exon 2 (lanes 3 and 4), GST-exon 3 (lanes 5 and 6), and no additional protein (lane 7) were added to the substrate-independent in vitro ubiquitination reaction master mix (MM) containing recombinant Uba1 (E1), recombinant cdc34, biotinylated ubiquitin, and ubiquitination buffer in the presence and absence of ATP and an ATP regenerating system, as described in Materials and Methods. The reaction was stopped after 90 min, and the reaction mixture was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. (B) Electrophoretically separated reaction mixtures containing the indicated GST fusion protein in addition to the master mix (lanes 8–13) or the master mix alone (lane 14) in the presence and absence of ATP and an ATP regenerating system were probed with a rabbit polyclonal antibody directed against GST. (C) cdc34 was precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 15–17) or the master mix alone (lane 18) in the presence of ATP and an ATP regenerating system. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. (D) GST or GST fusion proteins were precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 19–21) or the master mix alone (lane 22) in the presence of ATP and an ATP regenerating system by using glutathione Sepharose beads. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with a rabbit polyclonal antibody directed against cdc34. The dots to the right of high molecular weight bands in lanes 6 and 17 identify ubiquitinated proteins; G, GST; 2, GST-exon 2 chimeric protein; 3, GST-carboxyl-terminal domain of exon 3 fusion protein.

cdc34 with proteasomal subunits and that this interaction was expected to be higher in drug-treated than in untreated infected mediated in infected cells by wild-type ICP0 and was apparent cells. The procedures were as described in Materials and Meth- only when proteasomal function was inhibited by MG132. Also, ods. Electrophoretically separated proteins were probed with a all known classes of E3 ligases interact with E2 enzymes in some rabbit polyclonal antibody directed against ICP0 exon 2 (Fig. 4) manner (reviewed in ref. 25). To test the hypothesis that ICP0 or monoclonal antibody to exon 3 (data not shown). Significant interacts with cdc34, GST or GST-ICP0 chimeric proteins were levels of ICP0 degradation products were observed in neither the precipitated from the reaction mixture with glutathione Sepha- rose beads. The precipitates were electrophoretically separated in a denaturing polyacrylamide gel and reacted with an antibody directed against cdc34 (Fig. 3D). The results show that cdc34 coprecipitates from the reaction with GST-exon 2 but not with GST-exon 3 or GST (compare lane 20 with lanes 19 and 21). Thus, cdc34 specifically interacts with ICP0-exon 2, which con- tains the RING finger domain. Because GST-exon 3 promotes ubiquitination, it has to interact with cdc34 transiently or with low affinity. In many RING finger E3 ligases, the E3 RING finger domain binds the E2 (reviewed in ref. 25).

ICP0 Is Not Degraded in a Proteasome-Dependent Manner in Infected Cells. Inasmuch as both proteins targeted for degradation and components of the ubiquitin-proteasome degradation system are known to physically interact with proteasome complexes (re- viewed in ref. 36) and that some RING finger E3 ligases such as Fig. 4. ICP0 does not undergo proteasome-dependent degradation. HEL TRAF2 target themselves for proteasome-dependent degrada- fibroblasts infected with 10 pfu of HSV-1(F) (lanes 2–7) or R7914 (lanes 9–14) tion (37), we examined the effect of MG132 treatment on ICP0 per cell were mock-treated or treated with MG132 for 1 h before harvest at levels during infection of HEL cells exposed to 10 pfu of indicated times. The lysates were solubilized and subjected to electrophoresis HSV-1(F) or R7914 recombinant virus. If ICP0 were degraded in denaturing gels and reacted with a mouse monoclonal antibody against in a proteasome-dependent manner, the levels of ICP0 would be ICP0.

8818 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.161283098 Van Sant et al. Downloaded by guest on October 1, 2021 Fig. 5. Model of the shuttling of ICP0 between nucleus and cytoplasm. As discussed, the results of this and preceding studies from this laboratory suggest that ICP0 shuttles between nucleus and cytoplasm depending on the nature of the dynamic association of the protein with proteasomes. In the presence of MG132, ICP0 is sequestered by proteasomes in nuclei either early or late in infection. In the absence of the drug, ICP0 is retained primarily in the nucleus early in infection and in cytoplasm at midpoint and later times after infection.

presence nor the absence of MG132 in cells infected with Fig. 6. Models for E3 ubiquitin ligase function. E1 ubiquitin-activating HSV-1(F) and R7914 at any time point. We conclude that ICP0 enzymes are shown in dark blue, E2 ubiquitin-conjugating enzymes in green, was not degraded to a significant degree in a proteasome- RING finger subunits and domains in yellow, substrate-binding subunits and domains in magenta, and substrates in gray. (A) Multicomponent E3 complex dependent manner during the course of viral infection. modeled after SCF (adapted from ref. 40). The substrate is recruited to the Discussion complex by a specific substrate-binding domain within a protein that contains an F-box motif. The F-box interacts with SCF components Skp1 and Rbx1. This report sheds significant light on the involvement of ICP0 Recruitment of substrate p27 to SCF by the leucine-rich repeat (LRR) of the with the ubiquitin-proteasomal pathway. It is convenient to F-box protein Skp2 is depicted here. Other F-box proteins recruit different discuss the significance of key findings separately. substrates (reviewed in ref. 41). SCF contains 1 (CUL1), whereas other (i) ICP0 is dynamically associated with proteasomes and are components of other multicomponent E3s. The cullin serves as a remains bound to proteasomes in the presence of proteasomal scaffold to bind Skp1, the RING finger protein Rbx1, and the E2 cdc34, which inhibitor MG132. This observation is consistent with the report also interacts the RING finger. (B) RING finger unimolecular E3 ubiquitin ligase modeled after c-Cbl (adapted from ref. 25). The RING finger interacts with and published earlier that ICP0 colocalizes in dense spheroid-like allosterically activates the E2, UbcH4 (35). A Src-homology 2 (SH2) domain structures with proteasomal subunits in the cytoplasm of cells binds phosphotyrosine, such as platelet-derived growth factor receptor ␤ infected with the d120 mutant (23). These results also shed light (PDGF-R␤). (C) Proposed model for ICP0 unimolecular E3 ubiquitin ligase on the observation that at the midpoint of infection, i.e., after activity. Cdc34 binds the RING finger domain in ICP0-exon 2. Unidentified ICP0 is translocated to the cytoplasm, addition of MG132 causes substrates may bind exon 3, facilitating the transfer of ubiquitin from E2 to the the protein to be relocated to the nucleus (28). The results substrate. Also, it is possible that ICP0 E3 activity catalyzes regulatory self- support the hypothesis that at later stages of infection, ICP0 ubiquitination, which does not target ICP0 for degradation. shuttles between nucleus and cytoplasm and that in the presence of MG132, the shift in equilibrium to proteasome association causes a retention of ICP0 in the nucleus (Fig. 5). The location A caveat of this system is that it may not fully recapitulate the of ICP0 at the moment of capture, i.e., whether at the time of activity necessary to ubiquitinate a physiological substrate. Con- harvest and fixation of cells ICP0 is present in the nucleus or ceivably, a nonphysiologic function could be activated in vitro at MICROBIOLOGY cytoplasm may well depend on the composition of proteasomes, high nonphysiologic concentrations. The results nevertheless are and this may explain why in HEL fibroblasts ICP0 is found significant in that they point to two key features of ICP0. First, primarily in the cytoplasm after5hofinfection, whereas in exon 2 bound the E2 cdc34 but did not exhibit E3 ubiquitin ligase several human tumor cell lines the accumulation in the cyto- activity. Second, the E3 ubiquitin ligase activity was independent plasm begins at a much later time. of the RING finger domain encoded in exon 2. (ii) The association of the E2 protein cdc34 with proteasomes E3 ubiquitin ligases containing RING finger domains form was significantly increased in cells infected with wild-type virus two groups. The first (Fig. 6A) consists of E3 complexes such as but not in cells infected with the R7914 mutant and treated with SCF, which include a subunit with a RING finger domain that MG132. Furthermore, cdc34 formed several high molecular interacts with the cullin and E2 and a F-box protein that interacts weight bands consistent with ubiquitinated forms. The results with a specific substrate. The second (Fig. 6B) is a unimolecular indicate that in wild-type virus-infected cells, cdc34 is dynami- class of E3 enzymes, which contain a RING finger domain that cally associated with proteasomes in concert with and dependent binds the E2, whereas the substrate interacts with a different on wild-type ICP0 but not on ICP0 carrying the D199A substi- domain. c-Cbl is the prototype of this class. In contrast to HECT tution in the sequences encoded in exon 2. The latter observation suggested that this interaction is mediated by the sequences domain E3 enzymes, both unimolecular and complex RING encoding exon 2 of ICP0. Indeed, as shown in of Fig. 3D, cdc34 finger containing E3 enzymes do not appear to form a thioester binds the sequences encoded by ICP0-exon 2 in the presence intermediate with ubiquitin (reviewed in ref. 25). They bind of ATP. substrates and E2 enzymes and are thought to have a catalytic The E2 family is large and diverse, and the true range of E2 activity that activates E2 enzymes. By binding both the substrate proteins capable of interacting with ICP0 remains to be inves- and E2, the RING finger E3 functions primarily to tether and tigated. present the substrate to the activated E2 protein, thus facilitating (iii) In the substrate independent in vitro ubiquitination system the transfer of ubiquitin to the substrate. We propose that ICP0 used in these studies, ICP0 acted as an E3 ubiquitin ligase, and is a unimolecular E3 ubiquitin ligase as it promotes ubiquitin- this activity is encoded between codons 568 and 773 of exon 3. protein ligation and binds the E2 cdc34 (Fig. 6C).

Van Sant et al. PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8819 Downloaded by guest on October 1, 2021 The fundamental difference between c-Cbl and ICP0 E3 ICP0 E3 catalytic domain in exon 3, which stimulates E2 ubiquitin ligase activities is that, whereas c-Cbl RING finger ubiquitin conjugation activity. This would function to increase domain has intrinsic E3 activity (35), the RING finger domain the efficiency of ICP0 E3 ligase activity, because it would bring in ICP0-exon 2 does not. Moreover, mutations in the RING the E2 into the vicinity of the E3 catalytic domain, allowing it to finger domains of E3 enzymes Brca1, Siah-1, TRC8, NF-X1, kf-1, promote transfer of ubiquitin from the E2. In the substrate- and Praja1 or addition of zinc chelators that disrupt the RING independent in vitro ubiquitination system, the high concentra- finger domain block E2 binding and ubiquitin polymerization tion of reagents could ensure that random diffusion is sufficient promoted by these enzymes, suggesting interaction between the for GST-exon 3 to promote ubiquitin ligation from cdc34. We E3 RING finger domain and E2 is the seminal event in catalysis would expect a GST-fusion protein containing both domains to (38). Although Ubr1 and Rbx1 binding to E2s is not disrupted have increased E3 activity over GST-exon 3 in the in vitro system. by RING finger mutations, E3 activity is (reviewed in ref. 25). The colocalization of conjugated ubiquitin with ICP0 requires Thus, the RING finger might mediate E3 function by promoting the RING finger domain (21). Thus, although it is not required E2 catalytic activity, and the E2 might bind the E3 via other for in vitro E3 activity, the augmentation of E3 catalysis mediated subunits and͞or domains. In contrast, ICP0 exon 3 fragment by the RING finger domain may be required for meaningful E3 promotes ubiquitin-protein ligation in the complete absence of activity in vivo. the RING finger domain, showing that ICP0 intrinsic E3 activity All RING finger E3 ubiquitin ligases known to date have both is independent of the RING finger domain. This is unlike any E2-binding and ubiquitin ligation promotion activities, In some RING finger E3 hitherto described, as the RING finger domain E3 ligases, the two activities reside in the RING finger domains, is required for E3 activity in all known RING finger E3 ligases whereas in others, exemplified in this case by ICP0, only one of (reviewed in refs. 25 and 38). Known RING finger domains of these activities is localized in the RING finger domains, even proteins such as HdmX͞MdmX, which do not promote ubiquitin though the protein or complex expresses both activities. ligation, also do not have E3 ubiquitin ligase activity (39). Thus, These studies were aided by grants from the National Cancer Institute the catalytic mechanism of the ICP0 E3 ligase must be distinct (CA87761, CA83939, CA71933, and CA78766) of the United States from that of the other RING finger E3 ligases. Public Health Service. R.H. is a Howard Hughes Medical Institute The model we propose (Fig. 6C) is that E2 binding to the ICP0 Predoctoral Fellow. P.L. is a postdoctoral fellow of L’Association pour RING finger domain in exon 2 serves to tether the E2 to the la Recherche sur le Cancer (ARC, France).

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